This invention is generally in the area of the synthesis, purification, characterization and assay of combinatorial libraries of compounds.
Before the advent of combinatorial chemistry, the development of new biologically active compounds was dependent on the rational design and synthesis of compounds with a structure similar to existing biologically active compounds. Scientists looked at structure-activity relationships (SARs) to rationally develop a small series of compounds which might be expected to have similar bioactivity. The types of compounds selected for testing typically belonged to narrowly defined chemical classes, such as peptides, steroids, and the like. Such a process is generally known as a rationl approach to synthesis.
The rational approach involved selecting a singular molecular candidate, prepared either via chemical synthesis or isolated from natural sources, and evaluating the molecular candidate for a particular bioactivity. (See, for example, Testa, B. and Kier, L. B. Med. Res. Rev. 1991, 11, 35-48 and Rotstein, S. H. and Murcko, M. A. J. Med. Chem. 1993, 36, 1700-1710.) The cycle was repeated until a molecule possessing the desirable properties was identified. The rational approach was necessary because the synthesis, purification, and evaluation of potential lead compounds was time consuming, labor intensive, and expensive.
One relatively new approach for identifying biologically active compounds is known as combinatorial chemistry. In this approach, libraries of compounds with structures similar to the existing biologically active compound are prepared in small quantities, but in large numbers, and the entire library can be evaluated, for example, using various binding studies.
Many useful drugs have been discovered through the screening of randomly chosen compounds rather than through the rational approach. Combinatorial libraries of randomly-built chemical structures are routinely screened for specific biological activity. (Brenner, S. and Lerner, R. A. Proc. Natl. Acad. Sci. USA 1992, 89, 5381). Rapid screening allows one to evaluate combinatorial libraries, as well as large numbers of compounds, related or not, whether or not they are produced through chemical synthesis or isolated from natural sources, or by using the rational approach.
Both solid phase and solution phase chemistry have been used to develop combinatorial libraries. Solid phase combinatorial chemistry has been used, for example, to prepare oligonucleotide and peptide libraries. A limitation of solid phase chemistry is the scale at which it can be performed. Solution phase chemistry is applicable to a wider variety of chemical reactions, and is more amenable to scaleup, other than solid phase chemistry.
A limitation with combinatorial chemistry is not only in generating new compounds, but purifying, analyzing and screening the compounds. Often, so many compounds can be synthesized that a bottleneck is created at the purification, chemical analysis, and bioassay stages.
Several methodologies have been developed to synthesize, purify, analyze and screen combinatorial libraries. Some of these involve preparing compounds in multi-well plates, and others involve multi-tube arrays. A limitation of using multi-well plates is that it the scale of the reactions tends to be rather limited due to the size of the wells, and it can be difficult to determine chemical yield, since it is difficult to obtain the weight of individual samples in a plurality of wells. Synthesizing compounds in multi-tube arrays allows one to scale up the synthesis somewhat, but suffers from the disadvantage that the tubes are not fixed in position, so that human error can occur if the tubes are misplaced.
Further, there is little standardization in the combinatorial chemistry field. For example, multi-tube arrays often include 48 or 96 tubes. However, automated purification and chemical analysis equipment is not necessarily designed around the number of tubes in the multi-tube arrays used to prepare the compounds. This makes it difficult to track the identity and properties of compounds as they progress through stages of synthesis, purification and chemical analysis.
Prior attempts at overcoming these limitations have typically involved placing individual labels eg., bar codes on each tube in a multi-tube array. The bar codes allow one to keep track of tubes which might have been misplaced due to human error, and also to keep track of tubes moved to and from different multi-tube racks with varying numbers of tubes per rack.
An example of a combinatorial approach that uses bar codes to track the synthesis, purification and chemical analysis of libraries of compounds is a system developed by MDS Panlabs. This system synthesizes compounds at a 1 mmole scale in multi-tube arrays, subjects the compounds to preparative scale HPLC, and analyzes the compounds by flow inject mass spectrometry. Each tube in the array is identified with a bar code, and is moved from stages of synthesis, purification and chemical analysis using robotic arms. The bar codes are typically attached using an adhesive, which can evaporate at any stage when the tubes are subject to conditions involving increased temperatures and/or reduced pressures.
Given the size of many combinatorial libraries, it may be a tremendous burden to place, and keep track of, individual bar-codes on a plurality of tubes. It would be advantageous to provide new devices and processes for analyzing and screening large numbers of compounds without the need to generate bar codes for each tube used to synthesize, characterize or otherwise handle the compounds. The present invention provides such devices and processes.
The present invention is directed to devices and processes for synthesizing, purifying and analyzing large numbers of compounds, in particular, those generated for use in combinatorial and lead optimization libraries.
The processes involve generating a series of compounds in multi-well plates or multi-tube racks, storing the location of the individual tubes in a computerized device, transferring the contents of each tube to a chromatographic device for purification/characterization, removing the solvent from each eluted fraction containing the compound of interest, chemically analyzing the compounds, and then optionally submitting them for bioassays, while maintaining the ability to correlate the position of the tubes in the original multi-well plate or multi-tube array to the position of the tubes generated or used in the next stage of the operation.
The devices include one or more multi-tube arrays, a chromatographic or other purification device, means for transferring the contents of the tubes to the purification device, a solvent evaporator, means for adding solvent to the solvent-evaporated tubes, analytical instrumentation, and a means for transferring some or all of the contents to, and optionally from, the analytical instrumentation. Optionally the devices include a weighing instrument.
In one embodiment, the multi-tube array(s) include a cover, which may be placed on or removed from the array by computer control. This permits the computer to move tubes from the multi-tube array to other locations, such as a weighing station, and also protects the tubes from being moved manually. This overcomes the need to place a bar code on each tube to minimize the types of human error which result in misplaced tubes, and minimizes the error associated with evaporation of adhesives on the bar code label, which can often be greater than ten percent of the weight of the sample.
In some embodiments, the number of tubes in the multi-tube arrays in each step is constant. Alternatively, the number of tubes can vary from one stage to the next. However, a correlation between the orientation of the tubes from one stage can be made with the orientation of the tubes in a subsequent stage.
By correlating the orientation of tubes from one stage to the next, it is possible to use a single bar code or other identifying mark for each multi-tube array rather than for each individual tube. This simplifies data collection for large numbers of compounds.
Compounds which can be evaluated using the process and methods described herein include, for example, pharmaceutical compounds and agricultural chemicals, such as insecticides, pesticides, and herbicides.
In one embodiment, the compounds are arranged in the multi-tube arrays in the form of arrays of different chemical compounds with a common structure, and are each modified in a controlled fashion to create a combinatorial library of structurally related compounds. The common structure preferably has one, and more preferably, at least two sites capable of undergoing a reaction to change the structure, usually by the addition of other molecules.
In one embodiment, the compounds are not synthesized using the devices and processes described herein, but are purified, analyzed and optionally subjected to bioassays after having been previously prepared.
The steps described above can be carried out globally on the array or individually on each tube. For example, during the synthesis step, reagents can be added globally to each of the tubes. During the purification step, the contents of the tubes can be individually transferred to a purification device. When the solvent is removed from the tubes, the entire rack can laced in a solvent removal device. When the reaction products are characterized, the contents of each tube can individually be sent to analytical instruments. Depending on the particular device which is used, those of skill in the art can readily optimize the process by performing certain steps globally and certain steps individually on the tubes in the arrays.